Melting of Hemoglobin in Native Solutions as measured by IMS-MS.

Thermally induced structural transitions of the quaternary structure of the hemoglobin tetramer (human) in aqueous solution (150 mM ammonium acetate) were investigated using a variable temperature electrospray ionization (vt-ESI) technique in combination with ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements. At low solution temperatures (28 to ∼40 °C), a heterotetrameric (αβ) complex is the most abundant species that is observed. When the solution temperature is increased, this assembly dissociates into heterodimers (holo αβ forms) before ultimately forming insoluble aggregates at higher temperatures (>60 °C). In addition to the holo αβ forms, a small population of αβ dimers containing only a single heme ligand and having a dioxidation modification mapping to the β subunit are observed. The oxidized heterodimers are less stable than the unmodified holo-heterodimer. The Cys residue of the β subunit is the primary site of dioxidation. The close proximity of this post translational modification to both the αβ subunit interface and the heme binding site suggests that this modification is coupled to the loss of the heme and decreased protein stability. Changes in the charge state and collision cross sections of these species indicate that the tetramers and dimers favor less compact structures at elevated temperatures (prior to temperatures where dissociation dominates).